Ricin is a potent class B protein toxin found in castor seeds. Its ease of preparation has made it a favorite agent for domestic and international terrorists. Since vaccination against the toxin is not a plausible strategy for protection of the general populace, NIAID has made identification and production of small molecule inhibitors of ricin a top priority in its program for Biodefense. Toward this goal, our project is a collaborative effort from three laboratories with complimentary skills that facilitate inhibitor design, synthesis and testing. The project will be initiated with screens of existing commercial compounds to identify ricin inhibitors. Since the x-ray structure of ricin is known, it can serve as a template for a modern computer based virtual screen of large commercial libraries. The most attractive candidates will be assessed in an in vitro assay system. An initial screen of 1000 compounds has already identified three inhibitors including one, called Z3911, with an IC50 of 1 DM. In addition, high throughput screens using a newly developed micro-plate assay will be conducted at the NSRB facilities at Harvard. Inhibitors from both virtual and physical screens will then be optimized in design cycles using both structure-base methods and specifically created diversity libraries using state of the art combinatorial chemistry. We expect to improve IC50 values at least 10 to 100 fold into the nanomolar range. Our first effort to improve inhibitor strength is to diversify the Z3911 platform at three positions, creating a small library of derivative compounds. A detailed synthetic strategy is presented, and serves as an example for the manner in which additional leads will be developed from as yet unknown platforms. In the design scheme, reasonably strong in vitro inhibitors, like Z3911, will be tested early on in a Vero cell assay. This will allow us to assess bioavailability, and cell toxicity, and to restrain further design efforts towards useful compounds. Potent, non-toxic, ricin inhibitors will then be assessed in an animal challenge model for the final refinement of compounds. Our goal is create ricin (and homologous shiga toxin) inhibitors that can prevent, or alleviate toxicity in animals and serve as the basis for development, by commercial or government agencies, of true antidote drugs.